1. Field of the Invention
The invention relates generally to methods of culturing stem cells and progenitor cells.
2. Description of the Prior Art
Functional recovery following brain and spinal cord injuries and neurodegenerative diseases is likely to require the transplantation of exogenous neural cells and tissues, since the mammalian central nervous system (CNS) has little capacity for self-repair. However, neural cell or tissue transplantation is limited by the lack of tissue donors and the low survival rate of grafted cells. There is a need for an alternative strategy for building biological substitutes, such as a three-dimensional (3D) culture of neural cells to repair or replace the function of damaged nerve tissues. Tissue engineering that combines neural cells and polymer scaffolds may generate functional 3D constructs to serve as replacement tissues or organs (Bellamkonda, 1998; Woerly, 1999). Since neurons are not capable of proliferating and neurons in culture are short-lived, there remain significant challenges for neural tissue engineering. Recent advances in neural stem/progenitor cell biology show that progenitors can be isolated from the embryonic or adult CNS and placed in culture, where they are highly proliferative and differentiate into neurons and glial phenotypes (McKay, 1997; Ma, 1998; 2000; Gage, 1998; 2000; Maric, 2000; 2003). Therefore, CNS stem and progenitor cells have the potential to be a valuable source of specific neural cell types, which could be used for the neural tissue engineering (Gage, 1995; Fisher, 1997).
Polymer scaffolds play a critical role in neural tissue engineering, since neural progenitors and progeny like most other mammalian cells are anchorage-dependent and require the attachment to a solid surface (Ruoslahti, 1997). Among polymer scaffolds, hydrogels are attractive because of their highly porous and hydrated structure that allow cells to assemble spontaneously and become organized into a recognized tissue and permit the infusion of nutrients and oxygen into, and exit of waste products and CO2 out of the cells. Collagen is a biologically derived hydrogel, the major class of insoluble fibrous protein in the extracellular matrix (ECM). Neural progenitor cells isolated from embryonic rat CNS tissue rapidly proliferate and differentiate into neurons and astrocytes in type I collagen gels (O'Conner, 2000). In response to collagen and basic fibroblast growth factor (bFGF), the collagen-entrapped neural progenitor cells rapidly expand and differentiate spontaneously into excitable neurons and formed synapses (Ma, 2004). The collagen demonstrates a critical support for neuronal survival and synaptic activity (O'Conner, 2001; O'Shaughnessy, 2003). Such stem/progenitor cell-collagen constructs may be particularly useful as engineered neural tissue replacement for brain or spinal cord injury.
However, neural tissue engineering using the cell-collagen constructs remains a significant challenge. The cells in the constructs are typically short-lived due to the difficulty in exchanging oxygen and nutrients, which compromises cells and leads to cell death. Cell culture in a simulated microgravity environment offers two beneficial factors: low fluid shear stress, which promotes cell-cell contacts, and initiation of differentiating cellular signaling (Goodwin, 1993); and randomized gravitational vectors, which affect intracellular signal transduction and gene expression (Jessup, 1993). The aggregation-differentiation-promoting effects of the RWV culture conditions provide an excellent in vitro system to further differentiate bioengineered tissue-equivalents, such as skin (for review see Unsworth, 2000), cartilage (Freed, 1997), cardiac cells (Carrier, 1999), prostate (Margolis, 1999), kidney and liver fragments (for review see Unsworth, 2000). A rotating wall vessel (RWV) bioreactor was also used to form a rudimentary tissue-like structure from neural precursor cells (Low, 2001).